Effect of Antibiotics in The First Week of Life On Faecal Microbiota Development

Arch Dis Child Fetal Neonatal Ed: first published as 10.1136/archdischild-2021-322861 on 9 May 2022. Downloaded from http://fn.bmj.
com/ on October 28, 2022 at World Health Organisation

Original research
Effect of antibiotics in the first week of life on faecal

microbiota development
Emmy Van Daele ‍ ‍,1 Kim Kamphorst ‍ ‍,2,3 Arine M Vlieger ‍ ‍,3
Gerben Hermes ‍ ‍,1 Christian Milani ‍ ‍,4,5 Marco Ventura ‍ ‍,4,5 Clara Belzer ‍ ‍,1
Hauke Smidt ‍ ‍,1 Ruurd M van Elburg ‍ ‍,6 Jan Knol1,7
► Additional supplemental ABSTRACT

material is published online Background Infants are frequently exposed to WHAT IS ALREADY KNOWN ON THIS TOPIC
only. To view, please visit the ⇒ Up to 20% of neonates receive antibiotics
journal online (http://d x.doi. antibiotics (AB) in the first week of life for suspected
org/1 0.1136/a rchdischild- bacterial infections. Little is known about the effect of because of (suspected) early-onset neonatal
2021-322861). AB on the developing intestinal microbiota. Therefore, sepsis. In older infants, antibiotics have been
we studied intestinal microbiota development with and shown to disturb the intestinal microbiota,
For numbered affiliations see but studies in newborns with a developing
without AB exposure in the first week of life in term born
end of article.
infants. microbiota are limited.
Methods We analysed the faecal microbiota from ⇒ Feeding type and delivery mode also affect
Correspondence to
Professor Jan Knol, Nutricia birth until 2.5 years of age by 16S rRNA gene amplicon the microbiota, but their effect in relation to
Research BV, 3584 CT Utrecht, sequencing in a cohort with 56 term born infants, antibiotic exposure in the first week of life has
Utrecht, The Netherlands; exposed to AB in the first week of life (AB+) (AB for 2–3 not yet been studied.
jan.knol@d anone.com
days (AB2, n=20), AB for 7 days (AB7, n=36)), compared
(HINARI) - Group A. Protected by copyright.

WHAT THIS STUDY ADDS
RMvE and JK contributed with 126 healthy controls (AB-). The effects of AB and
duration were examined in relation to delivery and ⇒ Antibiotic exposure in the first week of life
equally.
feeding mode. affects microbiota development throughout the
Received 18 August 2021 Results AB+ was associated with significantly first year of life, with more profound deviations
Accepted 17 March 2022 in infants born at term exposed for 7 days
Published Online First increased relative abundance of Enterobacteriaceae at 3
weeks and 1 year and a decrease of Bifidobacteriaceae, compared with only 2–3 days.
9 May 2022
from 1 week until 3 months of age only in vaginally ⇒ Antibiotic exposure in the first week of life
delivered, but not in C-section born infants. Similar resulted in deviations in the faecal microbiota
deviations were noted in AB7, but not in AB2. After development of vaginally delivered infants but
AB, breastfed infants had lower relative abundance of not of C-section delivered infants compared
potentially pathogenic Enterobacteriaceae compared with their respective controls. This could be
with formula fed infants and recovered 2 weeks faster attributed to the difference caused by delivery
towards controls. mode itself, with C-section delivered infants
Conclusions AB exposure in the first week of life deviating from vaginal controls up to 1 month
alters faecal microbiota development with deviations of age regardless of antibiotic exposure.
in the relative abundance of individual taxa until 1 year HOW THIS STUDY MIGHT AFFECT RESEARCH,
of age. These alterations can have long-term health PRACTICE AND/OR POLICY
consequences, which emphasises the need for future
⇒ This study underlines the importance of early
studies aiming at restoring intestinal microbiota after AB
administration. cessation of antibiotics started at birth because
of the prolonged effect on the intestinal
microbiota and possible impact on health.
INTRODUCTION
During the first 1000 days of life, the intestinal eosinophilic esophagitis, infantile colic, inflamma-
microbiota impacts health in later life through tory bowel disease and obesity.7–11
the interdependent development of microbiota, In mice-based studies, AB exposure in the first
immune system, growth and cognitive func- week of life altered microbiota composition and
tion.1 2 After birth, the intestinal microbiota immune function, but gavage with mature untreated
© Author(s) (or their develops rapidly, driven by exposure to microbes microbiota, restored the perturbation and reduced
employer(s)) 2022. Re-use from maternal, environmental and dietary sources.3 the negative health effects.12 To understand the full
permitted under CC BY-NC. No
commercial re-use. See rights During this early development, the intestinal micro- impact in humans and develop restoration strate-
and permissions. Published biota is unstable and susceptible to perturbations gies, more knowledge is needed.
by BMJ. such as those caused by antibiotic (AB) exposure. Worldwide, up to 20% of all neonates are
These perturbations may have long- term conse- prescribed ABs because of (suspected) early-onset
To cite: Van Daele E,
Kamphorst K, Vlieger AM, quences on the developing microbiota and also neonatal sepsis, although in most cases, sepsis is
et al. Arch Dis Child Fetal on the developing immune system,4 growth5 6 and unconfirmed and ABs could be discontinued after
Neonatal Ed have already been associated with increased preva- 48–72 hours.13–15 More prolonged AB exposure can
2022;107:F603–F610. lence of asthma, allergies, coeliac disease, eczema, gradually reduce overall diversity16 and richness17
Van Daele E, et al. Arch Dis Child Fetal Neonatal Ed 2022;107:F603–F610. doi:10.1136/archdischild-2021-322861 F603
Arch Dis Child Fetal Neonatal Ed: first published as 10.1136/archdischild-2021-322861 on 9 May 2022. Downloaded from http://fn.bmj.com/ on October 28, 2022 at World Health Organisation
Original research
Figure 1 Overview of the collected questionnaires, faecal sampling points and the age categories based on the age (days) at sampling. N, number
of infants for which samples were available for a given time point. d(ays), m(onths) and y(ears) indicate the age category ranging from birth until
around 2 years of age.
of the neonate’s intestinal ecosystem. Additionally, AB type also the first year of life. Nine faecal samples were collected from
determines the microbial perturbation through specific mech- infants (figure 1). Until discharge from the hospital, faeces were
anisms of action and host interactions. Studies in infants have sampled from diapers by hospital staff and immediately frozen
been inconclusive,17–19 but indicated types with faster recovery at −20°C. Sampling continued at home by the parents, using
towards the microbiota composition of controls.20 Therefore, sampling instructions and freezer storage. After 1 year, parents
more comparative studies are needed between durations and delivered the samples to the clinic after transport on ice. A final
types in AB regimes to optimise AB administration.21 sample was taken around 2 years, stored in the home freezer and
In this study, we investigated the microbiota development collected by the study nurse. At the hospital, all samples were
in a subset of the Intestinal Microbiota Composition after AB stored at −20°C.
treatment in early life (INCA) cohort.22 The primary aim was
to investigate the impact of AB exposure in the first days of life

16S rRNA gene amplicon sequencing
on microbiota development during the first 2.5 years of life. DNA was extracted from a maximum of 200 mg of the homo-
Secondary aims were to examine (1) short (2–3 days) versus long genised faecal sample at GenProbio srl (Parma, Italy) with the
(7 days) AB administration, (2) different AB types and (3) the QIAamp DNA Stool Mini kit according to manufacturer’s
impact of feeding and delivery mode on AB perturbation. instructions (Qiagen Ltd, Strasse, Germany). Sequencing libraries
were prepared according to the 16S Metagenomic Sequencing
MATERIALS AND METHODS Library Preparation Protocol (Part No. 15 044 223 Rev. B—Illu-
Study design mina) at GenProbio srl (Parma, Italy). Minor adaptations to the
This prospective, observational study has been described previ- published protocol23 are noted in online supplemental file 1).
ously.10 22 To study the impact of feeding mode on AB-induced Sequencing resulted in ~44 934 (SD 17205) reads per sample.
deviations, we selected a subset of 182 infants with 1128 samples Data were processed using NG-Tax 2.0 on demultiplexed fastq
who were exclusively breast-fed (BF) or formula-fed (FF) in the files, using default settings.24 Taxonomy was assigned using
first 3 months of life. An overview of collected samples and their SILVA reference database V.128.25 Amplicon sequence variants
selection for analyses reported here is provided in online supple- (ASVs) were defined as unique sequence variants. Two synthetic
mental figure 1). All AB+ infants received gentamicin, which mock communities were sequenced as positive controls.26
was combined with penicillin (ABPen), amoxicillin (ABAMX) or
amoxicillin with clavulanic acid (ABAMC). Statistical analysis of the baseline characteristics
Baseline characteristics were calculated using R 3.6.127 and
Data and faeces collection tableone28 (table 1). Differences between groups were examined
Baseline characteristics such as birth mode were assessed through by using the Fisher exact test for the categorical variables and
hospital records. Feeding mode was reported monthly during analysis of variance (ANOVA) for continuous variables. Not
Table 1 Baseline characteristics of the INCA cohort subset included in this study
AB- AB+
AB2 AB7
‍ ‍ ‍ ‍ ‍ ‍ ‍ ‍
N 126 56 20 36
GA, weeks (IQR)* 39.4 40.4 40.05 40.55

(38.5, 40.4) (39.4, 41.1) (39.3, 41.0) (40.1, 41.2)
Birth weight, mean grams (SD) * 3478 (515) 3711 (484) 3734 (428) 3699 (518)
Birth weight for GA z-score (SD) 0.15 (1.2%) 0.39 (1%) 0.30 (1%) 0.57 (0.9%)
Sex (Female %) 58 (46%) 28 (50%) 13 (65%) 15 (42%)
Delivery mode (Vaginal %) 83 (66%) 41 (73%) 15 (75%) 26 (72%)
Exclusive breast feeding at 3 m (Yes %) 47 (37%) 23 (41%) 17 (47%) 6 (30%)
Additional AB 1–6 m (Yes %)*,** 10 (8.2%) 13 (24%) 1 (5%) 12 (34%)
Additional AB 7–12 m (Yes %) 30 (27%) 9 (19%) 2 (10%) 7 (24%)
Statistically significant differences are indicated with *p<0.05 compared with AB-, **p<0.05 compared with AB2.
AB+, infants who received AB during their first week of life. Birth weight for GA z-score is calculated according to the z-score formula.77 AB2: AB exposure for 2 to 3 days in the first week of life, AB7: AB exposure for 7
days.
AB, antibiotics; AB-, control infants; GA, gestational age; m, months.
F604 Van Daele E, et al. Arch Dis Child Fetal Neonatal Ed 2022;107:F603–F610. doi:10.1136/archdischild-2021-322861
Original research
normally distributed variables were tested with the Kruskal- reference group. Differences between AB groups were assessed
Wallis test and indicated by their median and IQR. Bonferroni per age interval using ANOVA in the vegan package.40
correction was performed to correct for multiple testing.
RESULTS
Bioinformatic and statistical analysis of the sequence data Baseline characteristics of INCA cohort subset
All analyses were performed in R 3.6.1.27 Samples were stratified The baseline characteristics differed between AB- and AB+
into 11 age-based and right-closed intervals for statistical anal- for gestational age, birth weight and additional AB exposure
ysis (figure 1). The Jenks Natural Breaks Classification (classInt between one to 6 months (table 1). Birth weight z-score (birth
package) was used to calculate the optimal ranges.29 Because of weight corrected for gestational age), however, was comparable.
increasing increments between sampling, the x-axis (age) was AB2 differed from AB7 with regard to additional AB exposure
log2 transformed for visualisation. between 1 and 6 months (5% vs 34%, respectively, p=0.019).
Alpha diversity (within sample diversity) was calculated at ASV Baseline characteristics were comparable between the AB type
level using Picante30 and Microbiome31 packages and following groups (online supplemental table S1).
metrics: Faith’s phylogenetic diversity,32 ASV richness, Shannon
and Inverse Simpson. All except Shannon diversity needed loga-
rithmic transformation to obtain normal distribution for one-
Antibiotic-induced alterations to intestinal microbiota
way analysis of covariance. Consecutive analyses were corrected development
for the baseline characteristic that differed significantly between AB exposure during the first week of life did not alter microbial
AB+ and AB- and between AB2 and AB7. alpha diversity between birth and 2.5 years (online supplemental
Temporal trends in relative abundance were visualised using figure 1). The temporal patterns of the four major phyla (95% of
local regression with locally estimated scatterplot smoothing the average relative abundance) were compared with univariate
using ggplot2. These relative abundances did not meet normality analyses (figure 2). Relative abundance of Proteobacteria was
requirements and were therefore compared using beta regres- high overall, during the first months of life. AB exposure further
sion with BetaReg33 per age interval. The effects of AB on increased this relative abundance at 1 month (mean 43.6% AB+,
31.5% AB-) and 1 year (mean 17.6% AB+, 6.5% AB-). Actino-

the relative abundance of phyla were modelled including and
excluding delivery mode, feeding mode and additional AB expo- bacteria peaked around 3 months, but AB exposure decreased
sure between one and 6 months. The optimal beta regression their relative abundance at 1 week (mean 6.3% AB+, 18.2%
models, based on Akaike and Bayesian information criteria, only AB-), 2 weeks (mean 8.4% AB+, 24.4% AB-), 1 month (mean
included AB exposure in the first week of life without additional 17.5% AB+, 27.2% AB-) and 3 months (mean 26.5% AB+,
terms. 34.4% AB-). The average relative abundance of Bacteroidetes
Beta diversity (between sample diversity) was calculated was stable at approximately 10% during the first year of life.
using pairwise Weighted (WU)34 and Unweighted UniFrac (UU) Firmicutes drastically increased in relative abundance towards
distances.35 WU takes the relative abundance of each ASV into 2.5 years. Both were unaffected by AB.
account, whereas UU uses presence or absence of ASVs. Pair-
wise UU and UW distance matrices were used to plot principle Effect of antibiotic duration on long-term microbiota
response curves (PRCs).36 PRC analysis is a redundancy anal- development
ysis for interpreting longitudinal data. It visualises multivariate Based on univariate statistics, the temporal trajectories of Acti-
responses in a repeated observation design.36 37 The method was nobacteria and Proteobacteria were most affected by AB. Based
designed to analyse the treatment effect over time compared on UU, ABs increased several members of the Enterobacteria-
with controls, as it can disentangle the time-dependent effects ceae and decreased Bifidobacteriaceae at one and 2 weeks (online
from other possible determinants.38 39 In this study, time was supplemental figure 2A). For WU, ABs impacted similar taxa at
displayed on the x-axis and the intestinal microbiota develop- 1 year (p<0.05) (figure 3A). These AB deviations were similar
ment was shown compared with AB- infants as the baseline in AB2 and AB7, but only the microbiota composition of AB7
Figure 2 Temporal trajectories of the relative abundance of the four main phyla in the developing intestinal microbiota from birth to 2.5 years of
age. Thick lines represent the average with shading showing the 95% CI. Differences between AB+ and AB- were calculated using beta regression for
each age category. * and vertical grey shading: difference in relative abundance (p<0.05), AB+, infants who received AB during their first week of life,
AB-, infants who did not receive AB during their first week of life, LOESS, locally estimated scatterplot smoothing.
Original research
Figure 3 WU-based PRC analysis is a special case of RDA for multivariate responses in a repeated observation design, which is applied here on the

longitudinal infant intestinal microbiota dataset from birth till 2.5 years of age. (A) The infants who did not receive antibiotics during the first week
of life (AB-), were compared as a baseline to the antibiotic exposed infants (AB+). Bacterial genera shown are the main drivers of the differences
between AB+ and AB-: taxa on the same side of baseline as the curve are linked to an increased relative abundance at that time point, opposite sides
indicate a decrease. (B) AB- was also compared as a baseline with the different antibiotic durations of 2–3 (AB2) or 7 days (AB7). Significance was
tested at the different time points using an ANOVA like permutation test (*p<0.05 compared with baseline AB-). Covariates that were controlled for
included additional AB exposure between the age of one and 6 months. AB+, infants who received AB during their first week of life; AB-, infants who
did not receive AB during their first week of life; AB2, AB exposure for 2–3 days in the first week of life indicated with light grey shading; AB7, AB
exposure for 7 days indicated with grey shading; ANOVA, analysis of variance; ASV, amplicon sequence variants; PRC, principal response curve; RDA,
redundancy analysis; WU, weighted UniFrac.
differed from AB- baseline (figure 3B and online supplemental associated with decreased Bifidobacterium relative abundance
figure 2B). in FF (1 month) (figure 4C), which occurred later than in BF
AB types had a different impact on the microbiota (online infants (2 weeks) (figure 4B). In turn, AB+ was associated with
supplemental figure 3A,B) with ABAMX not deviating from AB- increased relative abundance of Parabacteroides in BF, and with
baseline and ABPEN deviating at 1 week and 1 year with increased increased relative abundance of Enterobacteriaceae and Entero-
relative abundance of Enterobacteriaceae members in WU and coccus in FF infants.
UU. UU-based deviations between ABAMC and AB- baseline were
limited to week one and involved different ASVs compared with DISCUSSION
ABPEN. ABAMC affected WU in the long- term with increased In this prospective, observational INCA study, we examined the
Enterobacteriaceae and Enterococcaceae at 2 weeks and 1 month microbiota development after AB exposure during the first week
and also Bifidobacterium at 1 week and 2.5 years. of life and found perturbations in the faecal microbiota devel-
opment from 1 week until 1 year of age. These perturbations
Impact of delivery and feeding mode on AB-associated included decreased relative abundance of Bifidobacteriaceae
deviations in the faecal microbiota development while potentially pathogenic Enterobacteriaceae increased. This
Due to the relatively low number of faecal samples in the first study adds new insights into long-term compositional shifts after
week per feeding and delivery type (figure 1), effects were only neonatal AB exposure.16 41 Our results corroborate findings in
reported in samples collected between 1 week and 2.5 years. older infants with increased Enterobacteriaceae and decreased
Within AB-, microbiota deviated based on delivery mode from Bifidobacteriaceae after AB administration.16 20 42–44 Importantly,
1 week until 1 month (figure 4D). In vaginally delivered infants, the severity and duration of AB-mediated microbiota perturba-
the AB effect on the microbiota was still significant at 1 year tions increased with longer AB administration (5–7 vs 2–3 days).
with an increase of several Enterobacteriaceae, Enterococca- The results also align with a small study in preterm infants,
ceae and Streptococcaceae and decrease in Bifidobacterium and where >5 days AB exposure intensified perturbations compared
Escherichia-Shigella. In contrast, no AB- mediated deviations with 2–3 days.45
were noted between C-section born infants. Bifidobacteriaceae form a cornerstone in the early devel-
Compared with AB- BF baseline, AB+BF infants only deviated opment of the immune system. They were shown to promote
at 2 weeks, whereas AB+FF infants showed longer deviations B-cell maturation and associations with decreased inflammatory
from the first week up to 1 month (figure 4A). Because there responses and T-regulatory cell acquisition.46 Enterobacteriaceae,
was also a feeding effect during the first 6 months, the AB effect on the other hand, produce toxins and have lipopolysaccharides
was also analysed within the separate feeding groups. AB+ was on their outer membranes, which causes inflammation.47–49
Original research

Figure 4 WU-based PRC analyses in the different delivery and feeding mode groups. Bacterial genera shown are the main drivers for differences
between the groups and the baseline: positive effect on the curve is linked to increased ASVs in the positive spectrum and decrease of those in
the negative spectrum. (A) Breastfed controls (AB-BF) were compared as a baseline to antibiotic exposed (AB+) and FF infants. (B) Specifically
featuring the AB effect in breastfed children, with breastfed control children as a baseline (AB-BF) and (C) formula-fed children, with formula-fed
control children as a baseline (AB-). (D) Vaginally delivered control infants (AB-Vag) were compared as a baseline to antibiotic exposed (AB+) and
C-section delivered (Sectio) infants. Significance was tested at the different time points using an ANOVA like permutation test (*p<0.05). Delivery
mode analyses were controlled for feeding mode and vice versa. AB-, infants who did not receive AB during their first week of life; AB+, infants who
received AB during their first week of life also indicated within grey shading; ASV, amplicon sequence variants; BF, infants exclusively breast-fed in the
first 3 months of life; FF, infants exclusively formula-fed in the first 3 months of life; PRC, principal response curve; Sectio, infants delivered through
C-section, Vag, vaginally delivered infants; WU, weighted UniFrac.
Therefore, it is not surprising that reduced Bifidobacteriaceae, The long-term microbiota effect of ABs and its associated
often combined with an increase in potentially pathogenic negative health outcomes reinforce the need for implementing
Enterobacteriaceae, like Shigella, Klebsiella and Enterobacter, AB stewardship programs53–55 to avoid AB overuse.21 56 57 The
have been associated with immune- mediated disorders like microbiota perturbations were only significant after 5–7 days
asthma.50 Similar deviations were also found in functional disor- compared with 2–3 days AB which could explain previous find-
ders like infantile colic51 and irritable bowel syndrome.52 ings from the INCA cohort: namely higher incidence of infantile
Original research
colic, wheezing and food allergies in infants exposed for 5–7 days, represent relevant members of the intestinal microbiota such as
but not for 2–3 days.10 58 If this observed difference between 2–3 bifidobacteria more accurately, which makes them especially fit
days and 5–7 days exposure is the result of longer AB exposure or for analysing the intestinal microbiota of infants.23 For future
the result of a concomitant infection or inflammatory response is research, whole genome shotgun sequencing could be used to
yet unclear. The AB7 infants were treated because of suspected increase the accuracy of species and strain detection.73 74 Another
early onset sepsis (EOS). EOS is rare in term infants,59–61 but it limitation may be that environmental factors and maternal-infant
is difficult to distinguish from normal neonatal physiology after interactions could have been confounders, because AB+infants
birth, and laboratory tests cannot always reliably detect or rule were admitted to neonatal wards, whereas AB- infants stayed
out EOS.62 Because the consequences of delaying treatment are with their mothers on the maternity ward and were discharged
significant, on average 82 newborns without EOS are treated for earlier. Last, we did not have sufficient data on perinatal AB
each case.15 63–65 In our study population, only two of the 36 AB7 exposure and were therefore unable to correct for it, although
infants had a positive blood culture. The others were also treated it is questionable to what extent this confounder is important to
for 5–7 days because of elevated inflammatory markers or clin- take into account.75 76 Additionally, the study was not primarily
ical symptoms. Uzan-Yulzari et al showed that the association designed (and thus underpowered) to conclude on AB types.
between neonatal AB exposure and growth was independent of Nevertheless, our results suggest that ABAMX induced less pertur-
the neonatal infection state.6 This suggests that the differences bations as it did not result in any differences from AB- (online
in microbiota development after AB treatment in our study are supplemental figure 3B). The addition of the β-lactamase inhib-
more likely the result of the AB treatment duration itself than itor clavulanic acid (ABAMC) was associated with higher levels of
caused by a possible EOS. Our new findings emphasise the need bifidobacteria compared with other AB types, which supports
for microbiota restoration to minimise aberrant immune devel- an earlier finding in a single subject.20 Dedicated studies are,
opment. Suggested strategies include prebiotics, probiotics and however, needed to further elucidate the optimal regime with
synbiotics66 but also faecal transfers, which partially restored the the least microbial perturbations.
microbiota of mice exposed to AB for 7 days.12 In conclusion, AB exposure in the first week of life in term
In vaginally delivered infants, the AB effect was most born infants disturbed the microbiota up to 1 year, with more

pronounced with microbiota deviations in the second week of significant deviations after longer AB exposure (5–7 days). Both
life. C-section born infants, however, showed similar pertur- C-section delivery and AB administration in the first week of
bations regardless of AB exposure. After C-section, microbiota life are associated with deviant intestinal microbiota, but the
perturbations occurred due to reduced vertical mother-infant two combined are not associated with further deviation. Breast-
transmission of important intestinal microbes such as Bacteroides feeding was associated with reduced severity and duration of
and Bifidobacterium, while transmission of other microbes like perturbations compared with formula feeding. Our observations
skin and mouth bacteria increased,67 as well as due to maternal may help to elucidate why AB-exposed infants have more health
AB administration prior to cord clamping.3 C-section delivery problems. It may also support the development of preventive and
already showed decreases in Bifidobacterium spp and increases curative strategies after AB exposure to stimulate the growth of
in opportunistic pathogens from hospital environments like beneficial microbiota in order to prevent future health problems.
Enterobacter, Enterococcus and Klebsiella.68 This resembled
the AB effect, which might explain the lack of an additional Author affiliations
1
AB effect in C-section infants as these infants already lack the Laboratory of Microbiology, Wageningen University & Research, Wageningen, The
affected microbial groups from birth. Netherlands
2
Pediatrics, Amsterdam Gastroenterology, Metabolism & Nutrition, Amsterdam
Feeding also has a major impact on early life microbiota devel-
Reproduction & Development, Amsterdam UMC Locatie AMC, Amsterdam, The
opment.69 In our study, ABs in the first week of life perturbed the Netherlands
microbiota of both BF and FF infants, but potentially pathogenic 3
Pediatrics, St. Antonius Hospital, Nieuwegein, The Netherlands
4
Enterobacteriaceae only increased in FF infants. Moreover, AB Laboratory of Probiogenomics, Department of Chemistry, Life Sciences and
perturbations were still notable at 1 month in FF infants but Environmental Sustainability, University of Parma Department of Chemical Life
Sciences and Environmental Sustainability, Parma, Emilia-Romagna, Italy
only until 2 weeks in BF infants. Breastmilk probably aids resto- 5
Interdepartmental Research Centre "Microbiome Research Hub", University of
ration through components like human milk oligosaccharides Parma, Parma, Emilia-Romagna, Italy
and live bacteria, which stimulate the growth of bifidobacteria 6
Pediatrics, Amsterdam Gastroenterology, Metabolism & Nutrition, Amsterdam
and reduce (potential) pathogens.70 Reproduction & Development Amsterdam, Amsterdam UMC Locatie AMC,
The strengths of this study are the quantity of samples and Amsterdam, North Holland, The Netherlands
7
Nutricia Research BV, Utrecht, The Netherlands
long-term follow-up. This enabled the investigation of the inter-
play of AB with delivery and feeding modes. Moreover, the
Acknowledgements First of all, we want to thank the mothers and infants who
quantity of sampling points allowed for detailed and long-term participated in the INCA cohort for their time and effort. Second, we would like to
detection of AB-induced perturbations within individuals. This acknowledge the help of all the hospital staff in the recruitment and collection of
was relevant as AB impact was not uniform over time, suggesting data, and especially Carin Bunkers and Nicole Rutten for their support. Last, we
that limited sampling points could lead to misinterpretation. thank Marta Magnifesta at GenProbio srl (Parma, Italy) for performing the DNA
extraction and sequencing of the faecal samples.
Finally, the number of infants receiving additional courses of
AB in the first year was low in this cohort, thereby reducing an Contributors JK and RMvE contributed equally to this paper. EVD
conceptualized the research questions, wrote the manuscript, performed the
important confounding factor. analyses and data interpretation, wrote and revised the manuscript. KK provided
The methodology for profiling the intestinal microbiota, data and revised the manuscript. AMV conceptualised the original cohort study
which targeted the V3 hypervariable regions of the bacterial and provided a clinical outlook for data interpretation and writing and revised
16S rRNA gene, provides a cost-effective overview of bacterial the manuscript. GH conceptualized the research questions, provided support for
community composition, however, resolution at species level is the data analysis and revised the manuscript. CM performed data analysis and
revised the manuscript. MV performed data analysis and revised the manuscript.
limited, and amplification bias cannot be unequivocally ruled CB acquired funding and revised the manuscript. HS supervised the research
out.71 72 The applied Probio_Uni and Probio_Rev primers were activity and revised the manuscript. RMvE conceptualised the original cohort
validated and, compared with other primers, they seemed to study and provided a clinical outlook for data interpretation and writing and
Original research
revised the manuscript. JK is guarantor, supervised the research activity and 11 Kamphorst K, Van Daele E, Vlieger AM, et al. Early life antibiotics and childhood
revised the manuscript. gastrointestinal disorders: a systematic review. BMJ Paediatr Open 2021;5:e001028.
12 Niu X, Daniel S, Kumar D, et al. Transient neonatal antibiotic exposure increases
Funding This work was supported by JPI HDHL in conjunction with ZonMW and
susceptibility to late-onset sepsis driven by microbiota-dependent suppression of type
Danone Nutricia Research and grant number IM2015.
3 innate lymphoid cells. Sci Rep 2020;10:12974.
Competing interests This work and the PhD research by EVD was financed by a 13 Versporten A, Sharland M, Bielicki J, et al. The antibiotic resistance and prescribing
EU Joint Programming Initiative namely A Healthy Diet for a Healthy Life (JPIHDHL, in European children project: a neonatal and pediatric antimicrobial web-
http:// www.healthydietforhealthylife.eu/) inconjunction with ZonMW and Danone based point prevalence survey in 73 hospitals worldwide. Pediatr Infect Dis J
Nutricia Research. GH is a full full-time employee of Chr Hansen A/S since January 2013;32:e242–53.
2020. CB received a grant financed by JPI HDHL inconjunction with ZonMW and 14 Mukhopadhyay S, Eichenwald EC, Puopolo KM. Neonatal early-onset sepsis
Danone Nutricia Research. RMvE was an employee at Danone Nutricia Research till evaluations among well-appearing infants: projected impact of changes in CDC GBS
2020. JK is a full-time employee of Danone Nutricia. guidelines. J Perinatol 2013;33:198–205.
Patient consent for publication Consent obtained from parent(s)/guardian(s) 15 Fjalstad JW, Stensvold HJ, Bergseng H, et al. Early-Onset sepsis and antibiotic
exposure in term infants. Pediatr Infect Dis J 2016;35:1–6.
Ethics approval The INCA study was approved by the ethical board of the St. 16 Fjalstad JW, Esaiassen E, Juvet LK, et al. Antibiotic therapy in neonates and impact
Antonius Hospital in Nieuwegein, the Netherlands (registered as NCT02536560), on gut microbiota and antibiotic resistance development: a systematic review. J
and informed consent was obtained from both parents. Participants gave informed Antimicrob Chemother 2018;73:569–80.
consent to participate in the study before taking part. 17 Rooney AM, Timberlake K, Brown KA, et al. Each additional day of antibiotics is
Provenance and peer review Not commissioned; externally peer reviewed. associated with lower gut anaerobes in neonatal intensive care unit patients. Clin
Infect Dis 2020;70:2553–60.
Data availability statement Data are available on reasonable request. The data
18 Bennet R, Eriksson M, Nord CE. The fecal microflora of 1-3-month-old infants during
are available on request with the corresponding author.
treatment with eight oral antibiotics. Infection 2002;30:158-60.
Supplemental material This content has been supplied by the author(s). It 19 Parm U, Metsvaht T, Sepp E, et al. Impact of empiric antibiotic regimen on bowel
has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have colonization in neonates with suspected early onset sepsis. Eur J Clin Microbiol Infect
been peer-reviewed. Any opinions or recommendations discussed are solely those Dis 2010;29:807–16.
of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and 20 Korpela K, Salonen A, Saxen H. Antibiotics in early life associate with specific gut
responsibility arising from any reliance placed on the content. Where the content microbiota signatures in a prospective longitudinal infant cohort. Pediatr Res
includes any translated material, BMJ does not warrant the accuracy and reliability 2020:1–6.
of the translations (including but not limited to local regulations, clinical guidelines, 21 van den Anker J, Allegaert K. Rational use of antibiotics in neonates: still in search of

terminology, drug names and drug dosages), and is not responsible for any error tailored tools. Healthcare 2019;7:28.
and/or omissions arising from translation and adaptation or otherwise. 22 Rutten NBMM, Rijkers GT, Meijssen CB, et al. Intestinal microbiota composition after
Open access This is an open access article distributed in accordance with the antibiotic treatment in early life: the IncA study. BMC Pediatr 2015;15:204.
Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which 23 Milani C, Hevia A, Foroni E, et al. Assessing the fecal microbiota: an optimized ion
permits others to distribute, remix, adapt, build upon this work non-commercially, torrent 16S rRNA gene-based analysis protocol. PLoS One 2013;8:e68739.
and license their derivative works on different terms, provided the original work is 24 Poncheewin W, Hermes GDA, van Dam JCJ, et al. NG-Tax 2.0: a semantic framework
properly cited, appropriate credit is given, any changes made indicated, and the use for high-throughput amplicon analysis. Front Genet 2019;10:1366.
is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. 25 Yilmaz P, Parfrey LW, Yarza P, et al. The SILVA and "All-species Living Tree Project
(LTP)" taxonomic frameworks. Nucleic Acids Res 2014;42:D643–8.
ORCID iDs 26 Ramiro-Garcia J, Hermes GDA, Giatsis C, et al. NG-Tax, a highly accurate and
Emmy Van Daele http://orcid.org/0000-0003-0961-0481 validated pipeline for analysis of 16S rRNA amplicons from complex biomes.
Kim Kamphorst http://orcid.org/0000-0003-0310-6540 F1000Res 2016;5:5.
Arine M Vlieger http://orcid.org/0000-0002-3869-2896 27 R Core Team. R: a language and environment for statistical computing, 2019.
Gerben Hermes http://orcid.org/0000-0003-4314-9553 Available: https://www.r-project.org/
Christian Milani http://orcid.org/0000-0002-5062-3164 28 Yoshida K. tableone: Create ’Table 1’ to Describe Baseline Characteristics. R package
Marco Ventura http://orcid.org/0000-0002-4875-4560 version 0.11.1, 2020. Available: https://cran.r-project.org/package=tableone
Clara Belzer http://orcid.org/0000-0001-6922-836X 29 Bivand R. classInt: choose univariate class intervals, 2020. Available: https://cran.r-
Hauke Smidt http://orcid.org/0000-0002-6138-5026 project.org/package=classInt [Accessed 1 Jul 2021].
Ruurd M van Elburg http://orcid.org/0000-0003-0310-6540 30 Kembel SW, Cowan PD, Helmus MR, et al. Picante: R tools for integrating phylogenies
and ecology. Bioinformatics 2010;26:1463–4.
31 Lahti L, Shetty SA. Microbiome R package, 2019. Available: http://microbiome.github.
REFERENCES io
1 Wopereis H, Oozeer R, Knipping K, et al. The first thousand days - intestinal 32 Faith DP. Conservation evaluation and phylogenetic diversity. Biol Conserv
microbiology of early life: establishing a symbiosis. Pediatr Allergy Immunol 1992;61:1–10.
2014;25:428–38. 33 Cribari-Neto F, Zeileis A. Beta Regression in R. J Stat Softw 2010;34:1–24.
2 Robertson RC, Manges AR, Finlay BB, et al. The Human Microbiome and Child Growth 34 Lozupone CA, Hamady M, Kelley ST, et al. Quantitative and qualitative beta diversity
- First 1000 Days and Beyond. Trends Microbiol 2019;27:131–47. measures lead to different insights into factors that structure microbial communities.
3 Van Daele E, Knol J, Belzer C. Microbial transmission from mother to child: improving Appl Environ Microbiol 2007;73:1576–85.
infant intestinal microbiota development by identifying the obstacles. Crit Rev 35 Lozupone C, Knight R. UniFrac: a new phylogenetic method for comparing microbial
Microbiol 2019;45:613–48. communities. Appl Environ Microbiol 2005;71:8228–35.
4 Gensollen T, Iyer SS, Kasper DL, et al. How colonization by microbiota in early life 36 Van den Brink PJ, Braak CJFT. Principal response curves: analysis of time-dependent
shapes the immune system. Science 2016;352:539–44. multivariate responses of biological community to stress. Environ Toxicol Chem
5 Kamphorst K, Oosterloo BC, Vlieger AM, et al. Antibiotic treatment in the first week 1999;18:138–48.
of life impacts the growth trajectory in the first year of life in term infants. J Pediatr 37 Paliy O, Shankar V. Application of multivariate statistical techniques in microbial
Gastroenterol Nutr 2019;69:131–6. ecology. Wiley/Blackwell (10.1111), 2016.
6 Uzan-Yulzari A, Turta O, Belogolovski A, et al. Neonatal antibiotic exposure impairs 38 Fuentes S, van Nood E, Tims S, et al. Reset of a critically disturbed microbial
child growth during the first six years of life by perturbing intestinal microbial ecosystem: faecal transplant in recurrent Clostridium difficile infection. Isme J
colonization. Nat Commun 2021;12:443. 2014;8:1621–33.
7 Tamburini S, Shen N, Wu HC, et al. The microbiome in early life: implications for health 39 Choudhury R, Middelkoop A, Boekhorst J, et al. Early life feeding accelerates gut
outcomes. Nat Med 2016;22:713–22. microbiome maturation and suppresses acute post-weaning stress in piglets. Environ
8 Korpela K, Salonen A, Virta LJ, et al. Intestinal microbiome is related to lifetime Microbiol 2021;23:7201–13.
antibiotic use in Finnish pre-school children. Nat Commun 2016;7:10410. 40 Oksanen J, Blanchet FG, Friendly M. Package ‘vegan’ Title Community Ecology
9 Dierikx TH, Visser DH, Benninga MA, et al. The influence of prenatal and intrapartum Package Version 2.5-6, 2019.
antibiotics on intestinal microbiota colonisation in infants: a systematic review. J 41 Tapiainen T, Koivusaari P, Brinkac L, et al. Impact of intrapartum and postnatal
Infect 2020;81:190-204. antibiotics on the gut microbiome and emergence of antimicrobial resistance in
10 Oosterloo BC, van Elburg RM, Rutten NB, et al. Wheezing and infantile colic infants. Sci Rep 2019;9:10635.
are associated with neonatal antibiotic treatment. Pediatr Allergy Immunol 42 Korpela K, de Vos WM. Antibiotic use in childhood alters the gut microbiota and
2018;29:151–8. predisposes to overweight. Microb Cell 2016;3:296–8.
Original research
43 Tanaka S, Kobayashi T, Songjinda P, et al. Influence of antibiotic exposure in the early 61 Benitz WE, Achten NB. Finding a role for the neonatal early-onset sepsis risk
postnatal period on the development of intestinal microbiota. FEMS Immunol Med calculator. EClinicalMedicine 2020;19:100255.
Microbiol 2009;56:80–7. 62 Sharma D, Farahbakhsh N, Shastri S. Biomarkers for diagnosis of neonatal sepsis:
44 Navarro-Tapia E, Sebastiani G, Sailer S, et al. Probiotic supplementation during a literature review;31:1646–1659, 2017. Available: https://doi.org/101080/
the perinatal and infant period: effects on gut dysbiosis and disease. Nutrients 1476705820171322060
2020;12:1–42. 63 Kerste M, Corver J, Sonnevelt MC. Application of sepsis calculator in newborns
45 Zwittink RD, Renes IB, van Lingen RA, et al. Association between duration of with suspected infection. 29:3860–3865, 2016. Available: https://doi.org/103109/
intravenous antibiotic administration and early-life microbiota development in late- 1476705820161149563
preterm infants. Eur J Clin Microbiol Infect Dis 2018;37:475–83. 64 van Herk W, Stocker M, van Rossum AMC. Recognising early onset neonatal sepsis: an
46 Lim HJ, Shin HS. Antimicrobial and Immunomodulatory Effects of Bifidobacterium essential step in appropriate antimicrobial use. J Infect 2016;72 Suppl:S77–82.
Strains: A Review. J Microbiol Biotechnol 2020;30:1793–800. 65 Goel N, Shrestha S, Smith R, et al. Screening for early onset neonatal sepsis: NICE
47 Croxen MA, Finlay BB. Molecular mechanisms of Escherichia coli pathogenicity. Nat guidance-based practice versus projected application of the Kaiser Permanente
Rev Microbiol 2010;8:26–38. sepsis risk calculator in the UK population. Arch Dis Child Fetal Neonatal Ed
48 Vatanen T, Kostic AD, d’Hennezel E, et al. Variation in microbiome LPS immunogenicity 2020;105:118–22.
contributes to autoimmunity in humans. Cell 2016;165:842–53. 66 Sohn K, Underwood MA. Prenatal and postnatal administration of prebiotics and
49 Zeevenhooven J, Browne PD, L’Hoir MP, L’Hoir MP, et al. Infant colic: mechanisms and probiotics. Semin Fetal Neonatal Med 2017;22:284–9.
management. Nat Rev Gastroenterol Hepatol 2018;15:479–96. 67 Bäckhed F, Roswall J, Peng Y, et al. Dynamics and stabilization of the human gut
50 Zimmermann P, Messina N, Mohn WW, et al. Association between the intestinal microbiome during the first year of life. Cell Host Microbe 2015;17:690–703.
microbiota and allergic sensitization, eczema, and asthma: A systematic review. J 68 Shao Y, Forster SC, Tsaliki E, et al. Stunted microbiota and opportunistic pathogen
Allergy Clin Immunol 2019;143:467–85. colonization in caesarean-section birth. Nature 2019;574:117–21.
51 de Weerth C, Fuentes S, Puylaert P, et al. Intestinal microbiota of infants with colic: 69 Martin R, Makino H, Cetinyurek Yavuz A, et al. Early-Life events, including mode
development and specific signatures. Pediatrics 2013;131:e550–8. of delivery and type of feeding, siblings and gender, shape the developing gut
52 Pittayanon R, Lau JT, Yuan Y, et al. Gut microbiota in patients with irritable bowel microbiota. PLoS One 2016;11:e0158498.
syndrome-a systematic review. Gastroenterology 2019;157:97–108. 70 Le Doare K, Holder B, Bassett A, et al. Mother’s milk: a purposeful contribution to the
53 Ramasethu J, Kawakita T. Antibiotic stewardship in perinatal and neonatal care. Semin development of the infant microbiota and immunity. Front Immunol 2018;9:361.
Fetal Neonatal Med 2017;22:278-283. 71 Rintala A, Pietilä S, Munukka E, et al. Gut microbiota analysis results are highly
54 Araujo da Silva AR, Albernaz de Almeida Dias DC, Marques AF, et al. Role of dependent on the 16S rRNA gene target region, whereas the impact of DNA
antimicrobial stewardship programmes in children: a systematic review. J Hosp Infect extraction is minor. J Biomol Tech 2017;28:19.
2018;99:117–23. 72 Chen Z, Hui PC, Hui M, et al. Impact of preservation method and 16S rRNA
55 Ibrahim NA, Makmor Bakry M, Ishak S, et al. A review of antibiotic used in suspected hypervariable region on gut microbiota profiling. mSystems 2019;4. doi:10.1128/

early-onset neonatal sepsis from Malaysian perspective: which ones to choose and mSystems.00271-18. [Epub ahead of print: 26 02 2019].
how long to give? Asian J Pharm Clin Res 2019;12:529. 73 Ranjan R, Rani A, Metwally A, et al. Analysis of the microbiome: advantages of whole
56 Wagstaff JS, Durrant RJ, Newman MG, et al. Antibiotic treatment of suspected and genome shotgun versus 16S amplicon sequencing. Biochem Biophys Res Commun
confirmed neonatal sepsis within 28 days of birth: a retrospective analysis. Front 2016;469:967–77.
Pharmacol 2019;10:1191. 74 Jovel J, Patterson J, Wang W, et al. Characterization of the gut microbiome using 16S
57 Thaulow CM, Berild D, Blix HS, et al. Can we optimize antibiotic use in Norwegian or shotgun Metagenomics. Front Microbiol 2016;7:459.
neonates? A prospective comparison between a university hospital and a district 75 Dhudasia MB, Spergel JM, Puopolo KM, et al. Intrapartum group B streptococcal
hospital. Front Pediatr 2019;7:440. prophylaxis and childhood allergic disorders. Pediatrics 2021;147. doi:10.1542/
58 Kamphorst K, Vlieger AM, Oosterloo BC, et al. Higher risk of allergies at peds.2020-012187. [Epub ahead of print: 08 04 2021].
4-6 years of age after systemic antibiotics in the first week of life. Allergy 76 Dierikx T, Berkhout D, Eck A, et al. Influence of timing of maternal antibiotic
2021;76:2599–602. administration during caesarean section on infant microbial colonisation: a
59 Schrag SJ, Farley MM, Petit S, et al. Epidemiology of invasive early-onset neonatal randomised controlled trial. Gut 2021. doi:10.1136/gutjnl-2021-324767. [Epub
sepsis, 2005 to 2014. Pediatrics 2016;138. ahead of print: 21 Nov 2021].
60 Braye K, Foureur M, de Waal K, et al. Epidemiology of neonatal early-onset 77 Hoftiezer L, Hof MHP, Dijs-Elsinga J, et al. From population reference to national
sepsis in a geographically diverse Australian health district 2006-2016. PLoS One standard: new and improved birthweight charts. Am J Obstet Gynecol 2019;220:383.
2019;14:e0214298. e1-383.e17.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance
Supplemental material placed on this supplemental material which has been supplied by the author(s) Arch Dis Child Fetal Neonatal Ed
Van Daele E, et al. Arch Dis Child Fetal Neonatal Ed 2022;0:1–8. doi: 10.1136/archdischild-2021-322861
Supplementary Figure 1
Supplementary Figure 1. Alpha diversity in age categories from birth to 2.5 years of age. Alpha diversity at different age categories
of the antibiotic (AB+) exposed and non-AB infants (AB-) did not differ at any of the time points, using one-way Analysis of
Covariance (ANCOVA) corrected for additional AB exposure between one and six months of age, ASV: Amplicon sequence variants.
Supplementary Figure 2. Unweighted UniFrac (UU) -based Principal Response Curves (PRC) complementary to the PRC in Figure
3. (a) The infants who did not receive antibiotics during the first week of life (AB-), were compared as a baseline to the antibiotic
exposed infants (AB+). Bacterial genera shown are the main drivers of the differences between AB+ and AB-: taxa on the same
side of baseline as the curve are linked to an increased relative abundance at that time point, opposite sides indicate a decrease
(b) AB- was also compared as a baseline with the different antibiotic durations of 2 to 3 (AB2) or 7 days (AB7) .
Significance was tested at the different time points using an ANOVA like permutation test (* = p-value < 0.05 compared to baseline
AB-). Covariates that were controlled for included additional AB exposure between the age of one and six months. AB-: infants
who did not receive AB during their first week of life, AB+: infants who received AB during their first week of life also indicated
within grey shading, AB2: AB exposure for 2 to 3 days in the first week of life, AB7: AB exposure for 7 days in the first week of life,
ASV: Amplicon sequence variants, UU: unweighted UniFrac.
Supplementary Figure 3. Weighted (WU) (a) and Unweighted UniFrac (UU) (b) -based Principal Response Curves (PRC) comparing
the impact of different antibiotic types administered during the first week of life. The infants who did not receive antibiotics during
the first week of life (AB-), were compared as a baseline to the different types of antibiotics ABPEN, ABAMX and ABAMC.
Significance was tested at the different time points using an ANOVA like permutation test (* = p-value < 0.05 compared to baseline
AB-). AB-: infants who did not receive AB during their first week of life, ABPEN: antibiotic exposure in first week of life with
gentamicin and penicillin, ABAMX: gentamicin and amoxicillin, ABAMC: gentamicin with amoxicillin and clavulanic acid. ASV:
Amplicon sequence variants, UU: unweighted UniFrac, WU: weighted UniFrac
Supplementary file 1 – material and methods

Study design summary
In short, 436 infants born at term were recruited from maternity and neonatal wards of four
teaching hospitals in the Netherlands. Infants with suspicion of infection, who received a
combination of broad-spectrum ABs in their first week of life (AB+), and healthy, unexposed
controls (AB-) were included. Blood cultures were taken before AB exposure was started. In case
of a negative blood culture, combined with a low clinical suspicion of infection and low C-reactive
protein, ABs were discontinued after 2 to 3 days (AB2). Otherwise ABs were continued for 5 to
usually 7 days (AB7). Infants without sufficient follow up, less than 7 samples over the first three
years of age, were not included for this study.
Detailed material and methods:
Partial 16S rRNA gene amplicons were generated by using two consecutive PCR reactions. First,
the V3 region of the 16S rRNA gene was amplified using primers (Probio_Uni/Probio_Rev) with
adapters overhanging at the 5’ end 2. Thermal cycling conditions were as follows: 5 min at 95°C;
35 cycles of 30 s at 94°C, 30 s at 55°C and 90 s at 72°C; followed by 10 min at 72°C. The PCR
amplicons, which were validated and checked on size by electrophoresis on a 2200 Tape Station
instrument (Agilent Technologies, USA), were cleaned by magnetic Agencourt AMPure XP DNA
purification beads (Beckman Coulter Genomics GmbH). To enable multiplexing, unique
combinations of Tag barcodes (8 base pairs) were attached using a second PCR with the following
cycling conditions: 3 min at 95 °C; 8 cycles of 30 s at 95°C, 30 s at 55°C and 30 s at 72°C; followed
by 5 min at 72°C. Each PCR was performed with a Verity Thermocycler (Applied Biosystems, USA).
DNA obtained was purified by means of a magnetic purification step involving Agencourt AMPure
XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany). The DNA
concentration of the amplified sequence library was determined by a fluorometric Qubit
quantification system (Life Technologies, USA). Amplicons were diluted to a concentration of
4 nM in 10-μl quantities and combined to prepare the pooled final library. 16S rRNA gene
amplicon sequencing was performed using an Illumina MiSeq sequencer and MiSeq Reagent Kit
v3 chemicals (Illumina Inc., USA) at GenProbio srl (Parma, Italy) according to manufacturer’s
instructions.
1. Hoftiezer, L. et al. From population reference to national standard: new and improved
birthweight charts. Am. J. Obstet. Gynecol. 220, 383.e1-383.e17 (2019).
2. Milani, C. et al. Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-
Based Analysis Protocol. PLoS One 8, e68739 (2013).

Effect of Antibiotics in The First Week of Life On Faecal Microbiota Development

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Arch Dis Child Fetal Neonatal Ed: first published as 10.1136/archdischild-2021-322861 on 9 May 2022. Downloaded from http://fn.bmj.

com/ on October 28, 2022 at World Health Organisation

Effect of antibiotics in the first week of life on faecal

► Additional supplemental ABSTRACT

(HINARI) - Group A. Protected by copyright.

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GA, weeks (IQR)* 39.4 40.4 40.05 40.55

(HINARI) - Group A. Protected by copyright.

(HINARI) - Group A. Protected by copyright.

(HINARI) - Group A. Protected by copyright.

(HINARI) - Group A. Protected by copyright.

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Supplementary file 1 – material and methods

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